The long term goal of this project has been to determine the sequence and regulation of the molecular events that take place when cells attach and spread on extracellular matrices to form two distinct plasma membrane domains; one domain is attached to the matrix and the other is exposed to the extracellular milieu. Attachment and spreading of cells is involved in a number of biological phenomena including embryogenesis, blood coagulation, wound healing, and metastasis. We have been using HeLa cell attachment and spreading on collagen as a model to study this problem. Results obtained during the last funding period have led us to hypothesize the following: Cell-substrate attachment induces the exocytotic upregulation of collagen receptors. Concurrently, substrate induced clustering of three non-integrin type collagen receptors as well as B1 integrin collagen receptors drives the receptors to bind to the cytoskeleton. Receptor clustering also turns on a sequence of several second messengers and feedback mechanisms to initiate and optimize cell spreading. Our current objectives are to: (1) Determine whether the three HeLa cell non-integrin collagen receptors are a family of receptors different than the integrins. (2) Characterize the pathways responsible for producing the sequence of second messengers that initiate HeLa cell spreading on a gelatin substrate. HeLa cells exhibit three variables during cell spreading; rate of spreading, percentage of cells that spread, and the extent of spreading. The goal is not only to fully characterize the basic second messenger sequence regulating cell spreading but to also begin to determine if there are subsets of second messengers that could regulate the different variables of spreading. (3) Determine whether the three non-integrin and the B1 integrin collagen receptors in HeLa cells act independently, additively, or synergistically on the same or different second messengers to facilitate HeLa cell spreading. Lastly, we will begin work to determine if what we observe with the regulation of HeLa cell spreading, also occurs with more complex cells such endothelial cells.